Vet Pathol 43:541–544 (2006)
BRIEF COMMUNICATIONS and CASE REPORTS
Fetus in Fetu in a Harbor Seal (Phoca vitulina richardi): Histopathologic, Genetic,
and Toxicologic Analysis
E. L. BUCKLES, F. M. D. GULLAND, B. M. ALDRIDGE, T. S. GELATT, P. S. ROSS, M. HAULENA,
AND L. J. LOWENSTINE
Abstract. A young harbor seal (Phoca vitulina richardi), stranded on the coast of California, was
found to have a 20-cm-diameter cranial cervical mass. Surgical excision revealed the subcutaneous mass
to be covered in haired skin with multiple glabrous areas and structures resembling a jaw with tooth
buds, eyelids, and a tail. The mass deformed the host pup’s skull. Histologic examination revealed
a complete vertebra in the tail, teeth in the jaw, and areas resembling tongue and larynx. Class 1 MHC
sequences amplified by polymerase chain reaction from the mass and the host twin were identical. The
mass was diagnosed as a fetus in fetu, a rare congenital anomaly in which 1 conjoined twin is completely
enclosed in the body of the other twin. The host pup died, and no additional defects were found;
however, blubber levels of persistent organic pollutants were high. The cause of the congenital anomaly
in this pup is uncertain.
Key words: Congenital defect; contaminants; fetus in fetu; harbor seal; major histocompatibility
complex; Phoca vitulina richardi; twin.
Fetus in fetu is an extremely rare developmental
anomaly of humans in which 1 monozygotic twin is
completely incorporated into the other twin’s body.6,9
Diagnosis of this condition can be confusing, the major
differential being a mature teratoma.10 However, by
definition, the tissues of a true fetus in fetu are arranged
around a vertebral axis, whereas those of a teratoma are
less well organized. Here we report a cervical fetus in
fetu in a young harbor seal (Phoca vitulina richardi) and
genetic and toxicologic analyses conducted to investigate the etiology of the lesion.
A 2- to 3-month-old female harbor seal pup collected
from Pebble Beach, Monterey County, California
(36u589N, 121u959W), on 16 June 1999, was brought
to The Marine Mammal Center, Sausalito, California,
for treatment of a massive swelling around its neck.
The pup weighed 11.4 kg and was 75 cm long (nose
to tail). The seal was bright, alert, and vocal, but had
an elevated respiratory rate (10 per minute compared
with a normal of 5 per minute). On physical examination, the most striking abnormality was an approximately 30-cm-diameter subcutaneous swelling on
the ventral aspect of the neck (Fig. 1). The skin over
the ventral surface of the swelling was ulcerated,
presumably because of traumatic abrasion. Hematology
revealed leukopenia (total leukocyte count, 3.0 3 109/l,
with 80% neutrophils, normal range 5.9–24.6 3 109/l).3
Erythrocyte and serum biochemical parameters were
within the normal limits for this species.3 Radiography
revealed multiple amorphous calcified objects within
the mass, but aspiration of the mass yielded only
blood. The pup was anesthetized with propofol at
5 mg/kg and isoflurane 2–4% and was prepared for
surgery.
Surgical exploration of the neck region revealed
a large, multinodular, partially haired subcutaneous
structure (Fig. 2) extending along the ventral midline
from the intramandibular area to the sternum. The mass
was completely covered by a network of blood vessels
and partially adhered to the skeletal muscle. After
removal of the mass by blunt dissection, it became
apparent that the pup’s skull was flattened dorsoventrally, with the mandible, larynx, and trachea deviated
to the left, presumably because of pressure from the
mass (Fig. 3). The pup did not recover from surgery.
Detailed examination of the 1.3 kg, 20 3 16 3 10-cm
mass revealed it to be almost completely covered by
haired skin with interspersed glabrous areas. The hair
was light tan with numerous dark brown spots,
consistent with the coat color of a harbor seal. The
glabrous skin was variably pigmented, mottled pink and
brown. The roughly elliptical mass was characterized by
several protrusions resembling distinct anatomical
structures such as a tail, a palpebrum, and jaws with
tooth buds. On incision, a small vertebra, complete with
marrow, was present in the tail bud; bone supported the
primitive jaws, which contained tooth buds. No ocular
tissues were evident in association with the palpebral
area. The remaining mass was composed of irregular,
alternating cystic and solid regions of differing consistencies. Occasional mucous membrane-lined cystic areas
were filled with a green substance resembling meconium.
In addition, there were several islands of bone, including
a hollow, elliptical, bone-lined cyst that contained
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Brief Communications and Case Reports
Vet Pathol 43:4, 2006
Fig. 1. Subcutaneous mass; host harbor seal pup. Lateral view of the live host harbor seal pup (Phoca vitulina)
with the subcutaneous cervical mass (fetus in fetu) covered by intact host skin.
Fig. 2. Fetus in fetu; harbor seal. Ventral view of the fetus in fetu after removal from the ventral cervical
subcutis of the host seal pup. Note, on the right side of the photograph, the palpebrum between the tail-like
appendage (arrow) and the rudimentary jaw and toothlike structures (arrow head ).
Fig. 3. Skull and mandible; host harbor seal pup. Ventral view of the cleaned skull from the host pup showing
distortion and asymmetry (*), from compression by the cervical mass. Bar 5 1.0 cm.
Fig. 4. Tail-like appendage; fetus in fetu, harbor seal. Photomicrograph of the tail-like appendage from the
fetus in fetu in which cartilage, bone, and hematopoietic elements can be seen. HE. Bar 5 0.5 mm.
Vet Pathol 43:4, 2006
Brief Communications and Case Reports
islands of cartilage. Solid areas of tissue were composed
of fat; dense white connective tissue; and soft, pale pink,
more myxomatous tissue.
Histologic examination of the mass revealed it to be
covered in normal haired and glabrous skin, complete
with adnexae. The cystic structures were lined with
epithelium that ranged from squamous to low columnar.
Areas of bone throughout the mass and embedded in the
walls of 1 cyst were histologically normal. The tail
vertebra contained normal hematopoietic elements and
cartilage endplates (Fig. 4). The majority of the solid
regions of the mass were composed of adipose tissue and
vessels. The soft pink material consisted of ill defined,
poorly organized nervous tissue, including both gray
and white matter, as well as epithelium resembling
choroid plexus. One area resembled a rudimentary
tongue composed of skeletal muscle covered by serrated,
keratinizing, stratified, squamous epithelium with intracytoplasmic eosinophilic hyaline granules. A tubular,
cartilage-lined structure consistent with a larynx was
adjacent to the tongue. Examination of the rudimentary
jaw revealed several distinct developing teeth complete
with dental papillae, odontogenic epithelium, dentin,
enamel, and ameloblasts (Fig. 5). No other tissues, such
as hepatic or renal parenchyma, were identified.
No significant gross or histologic lesions other than
the flattened skull and the lateral deviation of the
mandible were observed in the host pup during
a complete postmortem examination (bone marrow of
the host pup was not examined). The reproductive tract
of the host pup was within normal limits for an infant
female. Skin from both the host pup and the mass were
submitted for genetic analysis. Genomic deoxyribonucleic acid was isolated by using standard techniques,4
and an approximately 300 base pair region of the a3
domain of the class I major histocompatibility complex
(MHC) was amplified by polymerase chain reaction
(PCR) by using degenerate class I MHC-specific
primers.4 After amplification, the PCR products were
separated and compared by using denaturing gradient
gel electrophoresis (DGGE). This technique has been
used for MHC-genotyping in other species.1 At least 9
bands representing the class I MHCa3 domains were
identified in each tissue sample. The separated class I
MHC PCR products were visually compared and
showed an identical banding pattern (data not shown),
providing strong evidence that the tissues from the host
pup and ventral cervical mass were genetically identical.
A complete sequence analysis of these PCR products
was not performed.
Blubber collected from over the sternum of the host
pup was analyzed for congener-specific polychlorinated
biphenyls (PCB) and dichlorodiphenyl trichloroethane
(DDT) by using high-resolution gas chromatography/
543
high resolution mass spectrometry and gas chromatography/low resolution mass spectrometry, respectively, as
part of a larger survey of contaminants in stranded
marine mammals. The host pup had high levels of both
PCBs (SPCBs, 33.7 mg/kg lipid weight) and DDTs
(SDDTs, 69.6 mg/kg lipid weight). PCBs were above the
range of levels observed in free-ranging harbor seal pups
of similar age sampled in British Columbia and
Washington State (SPCBs, 2.3–15.4 mg/kg lipid). Comparable SDDT values are not available for harbor seal
pups.
Nomenclature of developmental anomalies is complex
and often confusing. In general, these anomalous growths
are divided into 2 main categories: teratomas, which are
neoplasms arising from primitive germ cells of the fetus,
or conjoined twins, which result from either incomplete
fission during blastogenesis or fusion of 2 embryos in
utero.2 Anomalies in the latter category can be further
defined by the site of attachment of the 2 twins or else be
classified as a parasitic twin. A parasitic twin is
a conjoined twin that is incomplete, attached to, and
fully dependent for growth upon its nearly normal,
conjoined twin. Fetus in fetu is a rare type of parasitic
twin in which the incomplete twin becomes fully incorporated into the body of the larger sibling.9 Incidence
of such parasitic twins in humans is rare.6 Incorporation
of the parasite is thought to be the result of anastomosis
of the vitelline circulation.9 Because of the relationship
with the circulatory system, the most common site of
inclusion in human cases is the abdominal or retroperitoneal cavity; however, hepatic, scrotal, renal, adrenal,
and cranial sites have been reported.9 The results of the
MHC analysis in the present seal case suggest that the
conjoinment resulted from incomplete fission of identical
twins, with subsequent incorporation of one twin into the
body of the other.
Distinguishing between a well-differentiated teratoma
and a poorly differentiated fetus in fetu can be difficult.
Teratomas are derived from multiple primitive germ-cell
layers, are generally found along the midline of the body
or within the gonads, exhibit autonomous growth, and
can progress to malignancy.10 Because they arise from
undifferentiated cells, the tumors contain tissues foreign
to the area from which they arise, such as respiratory
epithelium, cartilage, renal glomeruli, nervous tissue,
and hematopoietic tissue. Fetus in fetu can also contain
these tissue elements and exhibit autonomous growth,
but this entity does not metastasize, and the internal
structures are arranged around an axial skeleton.9 The
presence of a vertebra indicates that the mass in this seal
had passed through the primitive streak stage of
embryogenesis. In the present case, the midline site of
origin and the poorly organized structure are consistent
with a cervical teratoma, but the existence of a clearly
r
Fig. 5. Tooth; fetus in fetu, harbor seal. Photomicrograph of a tooth from the primitive jaw area of the fetus in
fetu showing histologically normal development. HE. Bar 5 0.7 mm.
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Brief Communications and Case Reports
defined, well-developed vertebra warranted the diagnosis of fetus-in-fetu.
Harbor seals, like all pinnipeds, have a bicornuate
uterus; zonary, endotheliochorial placentation; and
a period of embryonic diapause in which implantation
of the blastocyst is delayed for 3 to 5 months after
fertilization.5 Twinning in marine mammals is rare, and
confirmed reports of live twin births in phocids are
currently confined to captive harbor seals and to
molecular evidence in wild Weddell seals (Leptonychotes
weddellii).7 Reports of conjoined twins in wild phocids
are rarer and previously limited to a report of conjoined
twin fetuses in a southern elephant seal (Mirounga
leonina).8 Conjoined twins have been observed in
a number of cetaceans, including striped dolphins
(Stenella coerueoalba), humpback whales (Megaptera
novaeangliae), sei whales (Balaenoptera borealis), and
minke whales (B. acutorostrata).8 These reports are all
based on data from fetuses found in utero in hunted
animals, so it is not known whether these twins would
have been viable. It is probable that anomalous twins
are rare in pinnipeds, because considerable numbers of
neonatal pinnipeds have been observed both in the field
and in rehabilitation centers after stranding. To our
knowledge, this is the first report of such an anomaly in
a harbor seal.
One interesting aspect of the present case is the high
level of PCBs and DDTs in the tissues of the host pup.
While these potentially teratogenic chemicals have been
heavily regulated in the USA and the industrialized
world since the mid-1970s, such agents have persisted at
high concentrations in many of the higher trophic levels
of wildlife populations. Although a variety of lesions in
seals are suggested to be associated with contaminant
exposure, no causal link can be made from these data,
and environmental teratogens to date have not been
investigated in production of parasitic twins in any
species. Whether or not the contaminants in this pup
were related to the developmental anomaly is a matter
for speculation.
Acknowledgements
We thank the staff and volunteers of The Marine
Mammal Center and C. J. McKinney-Sanderson for
assistance with this case; Howard Rhinehart for finding
cetacean twin literature; the Regional Dioxin Laboratory staff (Institute of Ocean Sciences) and Axys
Analytical Services (Sidney, B.C., Canada) for contam-
Vet Pathol 43:4, 2006
inant analysis; and the Arthur and Elena Court Nature
Conservancy for financial support for FMDG.
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Request reprints from Dr. Linda Lowenstine, Department of Pathology, Microbiology and Immunology,
School of Veterinary Medicine, One Shields Avenue,
University of California at Davis, Davis, CA 95616
(USA). E-mail: [email protected].